Erythropoietin (EPO) is a glycoprotein that stimulates erythroblast differentiation in bone marrow, thereby stimulating production of erythrocytes and increasing the circulation blood erythrocyte count. Human erythrocytes have a mean life of about 120 days, and therefore EPO is utilized to maintain the blood erythrocyte concentration to maintain an adequate level of red blood cells.
EPO's existence was first conceived in the early 1900's, but not definitively proven until sometime in the early 1950's through the work of Reissman and Erslev. See Carnot, et al., C.R. Acad. Sci. (France), 143:384-386 (1906); Carnot, et al., C.R. Acad. Sci. (France), 143:432-435 (1906); Reismann, Blood 5:372-380 (1950); and Erslev, Blood 8:349-358 (1953). It was determined that endogenous production of EPO is normally regulated by the level of tissue oxygenation. In general, hypoxia and anemia generally increase the production of EPO, which stimulates erythropoiesis. Normal plasma EPO levels range from 0.01 to 0.03 Units/mL and may see increases of 100 to 1000 fold during hypoxia or anemia events.
EPO functions by binding to and activating receptors on the surface of hematopoietic progenitor cells in the bone marrow. These cells serve to produce red blood cells and their activity rate is regulated through several hormones, including EPO. EPO serves to not only facilitate the production of red blood cells, but also serves as an anti-apoptotic chemical that modulates apoptosis in the progenitor cells.
When a body fails to produce sufficient red blood cells due to any number of reasons, the person is said to be anemic. Patients with chronic renal failure have impaired EPO production and this EPO deficiency is their primary cause of anemia. This failure is typically progressive and irreversible, requiring treatment with dialysis and/or other medications. A major breakthrough for these patients was the discovery of EPO and treatment with EPO.
EPO has excellent efficacy in the treatment of anemia and anemia derived from renal failure. See Eschbach, et al., N. England J. of Med. 316:73-38 (1987). However, while proven useful, the inability to produce EPO in significant quantities made its use severely limited. In the 1990's and 2000's the use of recombinant technology made it possible for obtain large amounts of proteins. U.S. Pat. No. 5,688,679 (to Powell), U.S. Pat. No. 5,547,922 (to Lin), U.S. Pat. No. 5,756,349 (to Lin), U.S. Pat. No. 4,703,008 (to Lin), and U.S. Pat. No. 4,677,195 (to Hewick et al.) provide guidance to manufacture of large scale production of recombinant EPO, each of which are incorporated by reference herein, in their entirety.
Recombinant human EPO (rHuEPO), or Erythropoietin Alfa, is a 165 amino acid glycoprotein manufactured by recombinant DNA. Egrie J C, et al., Immunobiol 1986; 72:213-224. Typically, the manufacture of rHuEPO is obtained by expression vector that comprises a gene coding for human EPO in CHO-, BHK-, or HeLa cell lines, by recombinant DNA technology or by endogenous gene activation. For example in any of the following patents: U.S. Pat. Nos. 5,733,761, 5,641,670, and 5,733,746, and international patent publication Nos. WO 93/09222, WO 94/12650, WO 95/31560, WO 90/11354, WO 91/06667 and WO 91/09955, and EP 1127104.
RHuEPO entered the market in 1989 when the FDA first approved its use for the treatment of anemia from chronic renal failure. As with other recombinant glycoproteins, the use of rHuEPO and its bioavailability is influenced by the glycosylation pattern of the recombinant glycoprotein. Two models, Chinese hamster ovary and baby hamster kidney (CHO and BHK-21) host cells have been thoroughly studied for their production of glycoforms that most closely resemble the natural human EPO forms. See Sasaki et al., J. Biol. Chem 1987; 262:12059-12076; Takeuchi et al., J. Biol. Chem 1988; 263:3657-3663; Nimitz et al., Eur. J. Biochem. 1993 213:39-56.
EPO and rHuEPO can be utilized to treat a number of patients, for example, those suffering from chronic renal failure, where rHuEPO has been shown to stimulate erythropoiesis in anemic patients with chronic renal failure. Eschbach J W, et al., NEJM 1998; 316:73-78, Eschbach J W, et al., Ann Intern. Med. 1989; 111:992-1000. Other uses for rHuEPO include treatment for HIV infected patients using Zidovudine, cancer patients on chemotherapy, for treatment of anemic patients scheduled for elective surgery, as well as adult and pediatric patients on dialysis.
While rHuEPO is able to be produced in large quantities, sufficient to provide for its use in medicinal settings world wide, it requires strict storage conditions of between 2 to 8° C. and has a shelf life of 2 years. Enhanced short-term stability dosage forms are also available, through an added preservative of benzyl alcohol, which is meant to control microbiological degradation. This short shelf-life is due to the fact that the production of the rHuEPO exhibits heterogeneity (such as aggregates, dimers and monomers) and impurities due to the manufacturing process. These aggregates, dimers, monomers and other residual impurities from the manufacturing process serve as catalysts in any number of reactions that promote the formation of dimers, aggregates or other sub- and/or non-potent forms. If the temperature is raised above the 2 to 8° C. range, these sub- and/or non-potent forms become apparent in liquid stability studies. The commercial EPO products also indicate that freezing the product is not an option (results in aggregation—loss of potency and potential rise in immunogenic response). The key is to extend the 2 to 8° C. stability timeframe. While isoforms are particularly relevant clinically, from a stability perspective, the lack of impurities, especially those that would tend to catalyze reactions leading to sub- and/or non-potent forms are particularly relevant here. Also relevant is the choice of buffer components and their interaction with the primary packaging—in this case, the vial and stopper.
Methods of isolation and purification of EPO are known in the art and include methods using anion and cation exchange, reverse phase HPLC, hydroxyapatite, hydrophobic interaction, affinity exclusion, size exclusion, as well as other filtration and purification techniques. See WO 2001035914, which is incorporated by reference in its entirety herein.
Each of the known purification processes have limitations in removal of certain impurities. Accordingly, there is a need to create new methods of purification of rHuEPO to facilitate a rHuEPO product having significantly reduced impurities, thereby allowing for enhanced stability and thereby greater shelf life for manufactured products.